Cell proliferation and programmed cell death play important roles in the growth and development of an organism. In proliferative diseases such as cancer, the processes of cell proliferation and/or programmed cell death are often perturbed. For example, a cancer cell may have unregulated cell division through either the overexpression of a positive regulator of the cell cycle or the loss of a negative regulator of the cell cycle, perhaps by mutation. Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator of apoptosis. Therefore, there is a need to develop new therapeutic agents that will restore the processes of checkpoint control and programmed cell death to cancerous cells.
RNA interference (RNAi) is an evolutionarily conserved process in which recognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptional suppression of gene expression. This suppression is mediated by short dsRNA, also called small interfering RNA (siRNA), which induces specific degradation of mRNA through complementary base pairing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function (see, e.g., Elbashir et al., Genes Dev., 15:188-200 (2001); Hammond et al., Nat. Rev. Genet., 2:110-119 (2001)). More recently, it was discovered that introducing synthetic 21-nucleotide dsRNA duplexes into mammalian cells could efficiently silence gene expression. Although the precise mechanism is still unclear, RNAi offers a new way to inactivate genes of interest. In particular, for the treatment of neoplastic disorders such as cancer, RNAi provides a potential new approach to modulate (e.g., reduce) the expression of certain genes, e.g., an anti-apoptotic molecule, a growth factor, a growth factor receptor, a mitotic spindle protein, a cell cycle protein, an angiogenic factor, an oncogene, an intracellular signal transducer, a molecular chaperone, and combinations thereof.
One such target is the polo-like kinase 1 (PLK-1) gene, which encodes a member of a family of serine/threonine protein kinases known as polo-like kinases (see, e.g., Nigg, Curr. Opin. Cell. Biol., 10:776-783 (1998)). In eukaryotes, the regulated progression through the cell cycle is controlled by a group of genes whose expression fluctuates throughout the cycle. Cyclin-dependent kinases and their associated regulatory subunits, the cyclins, are the primary regulators of the cell cycle. These heterodimeric complexes act by phosphorylating downstream targets that, in turn, trigger signaling events that liberate nuclear proteins necessary for entry into subsequent phases of the cell cycle. Polo-like kinases such as PLK-1 contribute to the activation and inactivation of these heterodimeric complexes.
As cells progress through the cell cycle, polo-like kinases undergo fluctuations in abundance, activity, and localization to control multiple stages of the cell cycle (Hamanaka et al., J. Biol. Chem., 270:21086-21091 (1995)). This family of kinases also functions in centrosome maturation (Lane et al., J. Cell. Biol., 135:1701-1713 (1996)), bipolar spindle formation (Golsteyn et al., J. Cell. Biol., 129:1617-1628 (1995)), DNA damage checkpoint adaptation (Arnaud et al., Chromosoma, 107:424-429 (1998)), and regulation of the anaphase-promoting complex (Kotani et al., Mol. Cell, 1:371-380 (1998)).
PLK-1 was the first member of this family of kinases to be identified as the mammalian counterpart to the Drosophila melanogaster gene polo, required for passage through mitosis (Golsteyn et al, J. Cell. Sci., 107:1509-1517 (1994); Hamanaka et al, Cell. Growth Differ., 5:249-257 (1994); Holtrich et al., Proc. Natl. Acad. Sci. U.S.A., 91:1736-1740 (1994); Lake et al., Mol. Cell. Biol., 13:7793-7801 (1993)). Expression of PLK-1 was shown to correlate with mitotic activity of cells (Golsteyn et al., J. Cell. Sci., 107:1509-1517 (1994); Lake et al., Mol. Cell. Biol., 13:7793-7801 (1993)) and to be high in tumors of several origins including lung, colon, stomach, smooth muscle, and esophagus (Holtrich et al., Proc. Natl. Acad. Sci. U.S.A., 91:1736-1740 (1994)). Overexpression or constitutive expression of PLK-1 has also been shown to induce malignant transformation of mammalian cells (Mundt et al., Biochem. Biophys. Res. Commun., 239:377-385 (1997); Smith et al., Biochem. Biophys. Res. Commun., 234:397-405 (1997)). Microinjection of PLK-1 antisense RNA into growing mouse NIH3T3 fibroblast cells was shown to block tritiated thymidine incorporation, suggesting that PLK-1 expression is restricted to and required by proliferating cells (Hamanaka et al., Cell. Growth Differ., 5:249-257 (1994)).
Further support for this conclusion is found in studies showing that elevated levels of PLK-1 expression are significant prognostic indicators of non-small cell lung cancer (Wolf et al., Oncogene, 14:543-549 (1997)), breast and lung cancer (Yuan et al., Am. J. Pathol., 150:1165-1172 (1997)), esophageal carcinoma (Tokumitsu et al., Int. J. Oncol., 15:687-692 (1999)), and squamous cell carcinomas of the head and neck (Knecht et al., Cancer Res., 59:2794-2797 (1999)). The pharmacological modulation of PLK-1 activity, expression, or function may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PLK-1 and investigative strategies aimed at modulating PLK-1 function have involved the use of antibodies and antisense oligonucleotides. For example, inhibition of PLK-1 expression using antisense oligonucleotides resulted in the loss of cell viability in cultured A549 cells and anti-tumor activity in nude mice A549 xenografts (Elez et al., Biochem. Biophys. Res. Commun., 209:352-356 (2000)). Similarly, U.S. Pat. No. 6,906,186 describes the inhibition of PLK-1 expression using antisense oligonucleotides in an in vitro cell culture system. However, these strategies are untested as therapeutic protocols and consequently there remains a long-felt need for agents capable of effectively inhibiting PLK-1 function in vivo.
Thus, there is a need for compositions and methods for specifically modulating PLK-1 expression. The present invention addresses these and other needs.